1. Area of the Art
This invention relates to xcex2-D-Glucuronidase substrates, methods of their synthesis and methods of their use.
2. Description of the Prior Art
The activity of xcex2-D-Glucuronidase (GUD) is extremely common in tissues of all vertebrates and many mollusks (Levvy, G. A. and Conchie, J., 1966, NY. p. 301). GUD is a key enzyme in the breakdown of proteoglycans constituting extracellular matrix and endothelial basal membrane. Yet, excessively high activity of the enzyme has been associated with certain tumors and bacterial infections (U.S. Pat. No. 4,351,823). For example, a metastatic potential of tumor cells has been linked with their GUD activity (M. Nakajima et al., J. Cellular Biochem. 36: 157-167, 1988). Similarly, elevated levels of CUD in gingival crevicular fluid and in saliva were indicative of existing periodontal disease and correlated with the likelihood of future disease progression (U.S. Pat. No. 6,063,588). In addition to its physiological importance, due to its smaller size and greater stability, GUD is a popular alternative to xcex2-galactosidase for use as an analyte indicator in enzyme assays and immunoassays. Consequently, the determination of the activity of GUD is of increasing importance in clinical chemistry, diagnostic assays, molecular biology, and certain environmental applications.
Conventionally, the determination of GUD involves the use of insoluble chromogenic substrates. For example, insoluble substrates 5-bromo-4-chloro-3-indolyl-xcex2-D-glucuronide (X-gluc) and indoxyl-xcex2-D-glucuronide were successfully used for the detection of E. coli on a solid media (U.S. Pat. No. 5,358,854). Similarly, in another study, E. coli colonies were identified by the formation of a black precipitate on chromogenic substrate 8-hydroxyquinoline glucuronide in combination with X-glucuronide (European Patent Application Publication 0025467). Although insoluble chromogenic GUD substrates provide a convenient and fast way to detect E. coli colonies based on their GUD activity, such insoluble substrates cannot be used in homogenous enzymatic assays. Quantitative enzyme assays are typically conducted in liquid extracts and rely on absorption or fluorescence detection methods. Consequently, soluble GUD substrates are required for such assays.
The detection of fluorescent molecules offers a very high signal-to-noise ratio because the incident excitation light does not impinge on the detection apparatus, and has a spectrum distinct and separable from that of the emission. For example, 3-carboxy-umbelliferyl xcex2-D-glucuronide, fluorescein glucuronide, resorufin glucuronide, methylumbelliferyl glucuronide were found to be useful substrates for fluorogenic assays of GUD activity (U.S. Pat. Nos. 4,226,978 and 5,599,670). However, because fluorescence emission is dependent on the intensity and wavelength of the excitation light, any factors in an assay mixture which affect the available excitation intensity or wavelength will correspondingly affect the apparent fluorescence output. Therefore, intrinsic background fluorescence common to biological samples is a serious problem limiting assay sensitivity. In order to minimize background fluorescence, the fluorescent compounds may be extracted prior to the sample analysis. However, such an approach makes the assay more time-consuming and complicated. Additionally, fluorometrical methods require a relatively expensive equipment.
Spectrophotometric assays, on the other hand, are very straightforward and moderately sensitive (Jefferson et al., 1986, Proc. Natl. Acad. Sci. USA 86:8447-8451). Because of the remarkable stability of GUD, one can enhance the sensitivity of spectrophotometric assays quite significantly by carrying out assays on a longer time scale to provide linear, reproducible results. Also, spectrophotometric assays are inexpensive, easy to automate, and easy to quantitate without sophisticated instrumentation (U.S. Pat. No. 5,599,670).
The currently preferred soluble substrate for the spectrophotometric measurement of GUD activity is p-nitrophenyl-xcex2-D-glucuronide. This substrate, when cleaved by GUD, releases the chromophore p-nitrophenol. At a pH greater than its pKa (around 7.15), the ionized chromophore absorbs light at 400-420 nm (xcexmax of 415 nm and xcex5=14,000), giving a yellow color. Unfortunately, often biological sample matrix is rich with compounds that absorb at the maximum wavelength of p-nitrophenol and, therefore, interfere with GUD detection.
Alternatively, phenolphthalein-xcex2-D-glucuronide may be used as a chromogenic substrate for GUD. The cleaved substrate by GUD phenolphthalein produces a purple color (xcexmax=550 nm and xcex5=3000) under alkaline conditions. This substrate is not in wide use now, due to its expense and very low extinction coefficient (U.S. Pat. No. 5,599,670).
Due to the above-discussed shortcomings of the related art, there is a need for new soluble GUD chromogenic substrates that allow fast, sensitive, and simple determination of GUD in a broad range of samples. Accordingly, it is an object of the present invention to provide such substrates, methods of their synthesis and methods of their use.
These and other objects and advantages are achieved in a xcex2-D-Glucuronidase (GUD) substrate of the following formula (IV): 
In this formula, R1, R2, and R7-R2 are independently selected from the group consisting of: hydrogen, fluorine, chlorine, bromine, iodine, alkyl, hydroxyl, alkoxy, carboxyl and nitro groups; R3-R6 are independently selected from the group consisting of hydrogen, fluorine, chlorine, bromine, iodine, nitro and amino; and M+ is selected from the group consisting of: proton, lithium, sodium, potassium, magnesium, calcium, barium, and ammonium ion. In one embodiment, R1-R8, R10, and R11 are hydrogen atoms, M+ is sodium, and R9 and R12 are chlorines.
Another aspect of the present invention provides a method of synthesizing a GUD substrate. The method comprises the steps of:
(a) forming a conjugate (III), having a formula: 
xe2x80x83and
(b) replacing Ac and Me groups with hydrogens.
In the formula (III), R1, R2, and R7-R12 are independently selected from the group consisting of: hydrogen, fluorine, chlorine, bromine, iodine, alkyl, hydroxyl, alkoxy, carboxyl and nitro groups; R3-R6 are independently selected from the group consisting of hydrogen, fluorine, chlorine, bromine, iodine, nitro and amino; M+ is selected from the group consisting of: proton, lithium, sodium, potassium, magnesium, calcium, barium, and ammonium ion; Ac is an acetyl group; and Me is a methyl group.
A further aspect of the present invention provides a method for determining the presence of GUD activity in a sample. The method comprises the steps of:
(a) contacting the sample with a GUD substrate (IV) of the present invention under a condition that allows the release of phenolsulphonphthalein;
(b) detecting phenolsulphonphthalein in the sample; and
(c) correlating the presence of phenolsulphonphthalein in the sample to a presence of GUD.
The detection step may be carried out by measuring the light absorption of the sample within a wavelength range that includes a wavelength of maximum absorption by phenolsulphonphthalein. In one embodiment of the present invention, the intensity of the measured light absorption is correlated to the activity of GUD in the test sample. In another embodiment, the intensity of the measured light absorption is correlated to the concentration of GUD in the test sample. The method may be used for both qualitative and quantitative analysis of GUD in samples.
The substrate of the present invention is well-suited for use with any detection equipment. In one embodiment, a colorimetric detector found on the CX(copyright), SX(copyright) and LX(copyright) SYNCHRON(copyright) systems (Beckman Coulter, Calif.) is used to detect phenolsulphonphthalein released from the substrate by GUD.
Another aspect of the present invention provides a test kit for determining the presence of GUD in a test sample. The test kit comprises GUD substrate (IV) of the present invention.
The most immediate application of the substrate and kit of the present invention includes a development of calorimetric assays for biochemical and clinical markers based on measured GUD activity in a sample. For example, Prostate Specific Antigen, myocardial infarction indicators, e.g. Creatine Kinase-MB and Troponin I, and traumatic muscle injury indicators, e.g. myoglobin, can be accurately detected using GUD substrate of this invention. Additionally, the detection and quantitative analysis of GUD activity in biological samples may allow diagnosis of certain bacterial infections.
Alternatively, recombinant DNA comprising a nucleic acid sequence encoding GUD, together with an appropriate controller sequence, may be introduced into a host cell or organism using any method known in the art, including transfection, transformation, infection, or microinjection to form a reporter gene system. In this application, the novel substrate of the present invention will be used to measure GUD activity and to correlate it to the activity of the controller element.
The present invention provides both economic and technical advantages over the use of other GUD substrates. The instant soluble GUD substrate provides highly sensitive, fast, and simple determination of GUD in a broad range of samples. Due to its high sensitivity, smaller quantities of the GUD substrate may be used.
The above-mentioned and other features of this invention and the manner of obtaining them will become more apparent, and will be best understood by reference to the following description, taken in conjunction with the accompanying drawings. These drawings depict only a typical embodiment of the invention and do not therefore limit its scope.